The long range goal of the proposed research is to understand the cellular and molecular mechanisms whereby opiates inhibit voltage-dependent calcium (Ca++) channels in mammalian neurons and to determine how this regulation relates to the ability of opiate analgesics to depress neurotransmitter release. This information is being sought, because it is through this presynaptic inhibitory action that opiates modulate the transmission of nociceptive information and exert their analgesic effects. The principal objectives of the research are to characterize the inhibitory coupling between opioid receptors and Ca++ channels in the nerve terminal and neuronal cell body with respect to the type and exocytotic function of the channels that are modulated and to determine the functional importance of alterations in voltage-dependent Ca++ signaling in mediating opioid inhibition of exocytotic release. The experiments will utilize whole-cell patch clamp techniques to record Ca++ currents in combination with monitoring of exocytosis using time-resolved measurements of membrane capacitance (Cm) in isolated peptidergic nerve endings and somata (currents only) of the hypothalamo-neurohypophysial system and in dissociated dorsal root ganglion (DRG) sensory neurons from adult rat. Using pharmacological blockers selective for specific types of Ca++ channels, together with specific voltage clamp protocols, we will determine whether the pattern of inhibitory coupling of opioid receptors to Ca++ channels expressed in supraoptic nucleus (SON) neurons parallels that for exocytotic channels in nerve endings that regulate vasopressin or oxytocin release. The relationship between opioid blockade of voltage- dependent Ca++ influx and inhibition of release will then be assessed by quantifying the effects of particular Ca++ channel antagonists and controlled alterations in [Ca++]i, measured with Fura-2, on the ability of kappa- or micro-opioid agonists to inhibit Ca++ currents and attenuate Cm responses in nerve ending and cell body preparations. In extending the analysis to functionally defined subsets of DRG neurons, we will determine the role of modulation in Ca++ influx and alterations in downstream components of the exocytotic process in mediating opioid inhibitory effects on the transmission of nociceptive signals. The proposed research, in contributing to a mechanistic understanding of opioid regulation of neurotransmission, should aid in the design of novel analgesic agents devoid of the tolerance or abuse liability characteristic of opiate drugs.